Pancreatic beta-cells synthesize large quantities of insulin. Growing evidence indicates that any of a number of deficiencies in insulin biosynthesis (genetic, or acquired) can lead to diabetes. We know that insulin biosynthesis begins with translation of preproinsulin. This short-lived precursor must be translocated into the endoplasmic reticulum (ER), signal peptide excised, and proinsulin properly folded in order to undergo successful export from the ER for delivery to the distal secretory pathway in which proinsulin-to-insulin processing and insulin storage in secretory granules finally occurs. In contrast, unsuccessful molecules may be degraded before they are even translocated into the ER, or may be restrained from anterograde export from the ER ? indeed, strong evidence indicates that misfolded proinsulin molecules are targeted for degradation. Secretory pathway protein degradation also involves other endogenous substrates that contribute to the differentiated pancreatic beta cell phenotype. The competing continuation of this multi-P.I. R01 will help clarify how three major mechanisms of secretory pathway protein disposal ? pre-translocation degradation; ER-Associated Degradation (ERAD); and Autophagy ? are all critical for proper beta-cell function. This proposal continues the longstanding association of three tightly collaborative investigators (Qi, Tsai, Arvan) that are experts in exactly these processes: preproinsulin translocation into the ER lumen with the subsequent folding/misfolding of proinsulin, secretory pathway protein degradation via ERAD, and an ER-to-lysosome degradative pathway that we believe is primarily ER-autophagy (ER-phagy). We have strong reason to believe that defects in these quality control mechanisms are linked to type 2 diabetes (T2D) as a result of insulin insufficiency, and this belief is supported by preliminary data. In this proposal, we seek to examine three interlinked areas related to the early secretory pathway of pancreatic beta-cells. For one, we will pursue studies in which infidelity of preproinsulin translocation across the ER membrane is directly linked to deficient proinsulin and insulin biosynthesis, leading directly to diabetes. Second, we will follow-up on some remarkable preliminary data demonstrating that de-differentiation of pancreatic beta-cells is triggered by a loss of efficient ERAD function, also leading directly to insulin-deficient diabetes. Finally, we not only delve deeply into the ER factors that trigger ER-phagic degradation of misfolded proinsulin, but we also propose a deeper understanding of how ineffective or improper ER-phagy can trigger beta cell failure, which also leads directly to insulin-deficient diabetes. These new research directions lead us to pursue a novel therapeutic approach to beta-cell secretory pathway dysfunction focused on stimulating intracellular protein clearance mechanisms, in order to prevent diabetes onset and/or limit its progression.